A translocation signal for delivery of oomycete effector proteins into host plant cells

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Bacterial1, oomycete2 and fungal3 plant pathogens establish disease by translocation of effector proteins into host cells, where they may directly manipulate host innate immunity. In bacteria, translocation is through the type III secretion system1, but analogous processes for effector delivery are uncharacterized in fungi and oomycetes. Here we report functional analyses of two motifs, RXLR and EER, present in translocated oomycete effectors2. We use the Phytophthora infestans RXLR-EER-containing protein Avr3a as a reporter for translocation because it triggers RXLR-EER-independent hypersensitive cell death following recognition within plant cells that contain the R3a resistance protein4,5. We show that Avr3a, with or without RXLR-EER motifs, is secreted from P. infestans biotrophic structures called haustoria, demonstrating that these motifs are not required for targeting to haustoria or for secretion. However, following replacement of Avr3a RXLR-EER motifs with alanine residues, singly or in combination, or with residues KMIK-DDK—representing a change that conserves physicochemical properties of the protein—P. infestans fails to deliver Avr3a or an Avr3a–GUS fusion protein into plant cells, demonstrating that these motifs are required for translocation. We show that RXLR-EER-encoding genes are transcriptionally upregulated during infection. Bioinformatic analysis identifies 425 potential genes encoding secreted RXLR-EER class proteins in the P. infestans genome. Identification of this class of proteins provides unparalleled opportunities to determine how oomycetes manipulate hosts to establish infection.

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Figure 1: Recognition of Avr3a by R3a follows delivery into the host cell by the T3SS of Pectobacterium atrosepticum (Pba).
Figure 2: Replacement of RXLR-EER motifs with alanine residues, singly or in combination, or with amino acids KMIK-DDK, prevents delivery of Avr3a into host cells.
Figure 3: Avr3a is secreted from haustoria and is translocated into the host cell in an RXLR-EER-dependent manner.


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This work was supported by grants from the Scottish Executive Environment and Rural Affairs Department (SEERAD) to S.C.W., P.R.J.B. and I.K.T. J.G.M. is supported by the Universidad Nacional de Colombia sede Palmira Agricultural Sciences department and the European Union Alban Programme. P.v.W. is supported by The Royal Society; L.M. is supported by a Commonwealth Scholarship and Fellowship Plan. The authors thank G. Cowan, H. Liu and E. Venter for technical assistance.

Author Contributions S.C.W., P.R.J.B., P.v.W. and I.K.T developed the concept and designed experiments. S.C.W. and S.G. performed P. infestans transformations and plant inoculations. P.C.B. carried out confocal microscopy and advised on cell biology. S.C.W. performed GUS assays and light microscopy. A.O.A. and J.G.M. quantified gene expression. Antibody detection of tagged transformants was performed by I.H. and S.C. L.M., J.G.M., E.M.G. and M.R.A. carried out experiments with P. atrosepticum. L.P. conducted all bioinformatics analyses.

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Correspondence to Stephen C. Whisson or Paul R. J. Birch.

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Supplementary information

Supplementary Information

The file contains Supplementary Figures S1 – S7 and Legends; Supplementary Methods and Supplementary Tables S1 – S5. Figure S1 summarizes the main findings of the paper. Figure S2 and Table S1 show gene expression profiles for P. infestans RxLR-EER genes, and figures S3-S7 and tables S2-S3 support RxLR-EER mediated translocation of Avr3a inside plant cells. (PDF 2025 kb)

Supplementary Information

The file contains Supplementary Bioinformatics Analyses: Identification of candidate RXLR proteins in the Phytophthora infestans draft genome sequence, and in the P. sojae and P. ramorum genome sequences, and design of conservative RXLR motif substitutions. Hidden Markov Model and Heuristic predictions of RxLR-EER proteins from oomycete genomes. (PDF 1186 kb)

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Whisson, S., Boevink, P., Moleleki, L. et al. A translocation signal for delivery of oomycete effector proteins into host plant cells. Nature 450, 115–118 (2007) doi:10.1038/nature06203

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